Synergistic Microbicidal Combinations

ABSTRACT

Antimicrobial compositions based on the synergistic combination of 2-methyl-3-isothiazolone and selected commercial microbicides where the compositions are substantially free of halogenated 3-isothiazolone, are disclosed. Particularly preferred are combinations of 2-methyl-3-isothiazolone together with benzoic acid, citric acid, sorbic acid, 1,2-dibromo-2,4-dicyanobutane, 1,3-dimethylol-5,5-dimethylhydantoin, phenoxyethanol, benzyl alcohol, zinc pyrithione or climbazole, that provide enhanced microbicidal efficacy at a combined active ingredient level lower than that of the combined individual 3-isothiazolone and commercial microbicide effective use levels.

BACKGROUND

This invention relates to synergistic microbicidal combinations and, inparticular, to synergistic microbicidal combinations of2-methyl-3-isothiazolone with one or more selected commercialmicrobicides, where the resulting composition is substantially free ofhalogenated 3-isothiazolone.

Kathon™ CG biocide (3/1 ratio of 5-chloro-2-methyl-3-isothiazolone to2-methyl-3-isothiazolone) and Neolone™ 950 bactericide(2-methyl-3-isothiazolone) are highly effective preservatives and may beused in combination with a variety of commercially availablepreservatives to prevent microbial contamination in personal careapplications, such as cosmetics and toiletries (see “Kathon™ CG Cosmeticand Toiletry Preservative” Bulletin CS-663, September 1997, and“Neolone™ 950 Preservative for Personal Care” Bulletin CS-707, May 2001,Rohm and Haas Company, Philadelphia, Pa.). U.S. Pat. No. 5,591,759discloses the use of 1,2-dibromo-2,4-dicyanobutane to stabilizeisothiazolone mixtures of5-chloro-2-methyl-3-isothiazolone/2-methyl-3-isothiazolone (9/1 ratio).Patent application WO 01/50855 discloses preservative formulations forextending the lifetime of cut flowers based on preservative formulationscontaining acrylic copolymer additives, saccharide derivatives andantimicrobial mixtures, including those based on2-methyl-3-isothiazolone in the presence of citric acid/citrates.

Many other microbicidal agents are known. These are commerciallyavailable for the control of microorganisms in a variety of loci.Sometimes, many microbicides cannot provide effective antimicrobialcontrol even at high use concentrations due to weak activity againstcertain microorganisms. Without effective microorganism control, loss ofproduct, inferior product, production time loss, health hazard and otherproblems may occur in the locus treated. There is a need for a method tocontrol various microorganisms that does not rely on high use levels ofcombinations of different microbicidal agents, but still provideseffective overall control of the microorganisms that is both quick andlong lasting.

The problem addressed by the present invention is to overcomedeficiencies of previous microbicide combinations by providing acombination of microbicidal agents that is more effective than theindividual microbicides used alone and that can be used at lower overalllevels while providing efficacy similar to the original individualmicrobicide levels.

STATEMENT OF INVENTION

The present invention provides a microbicidal composition comprising asynergistic mixture, the first component of which is2-methyl-3-isothiazolone, and the second component of which is one ormore commercial microbicides selected from the group consisting ofbenzoic acid, sorbic acid, 1,2-dibromo-2,4-dicyanobutane,1,3-dimethylol-5,5-dimethylhydantoin, phenoxyethanol, zinc pyrithioneand climbazole; wherein the ratio of the first component to the secondcomponent is from 1/0.001 to 1/1000; and wherein the composition issubstantially free of halogenated 3-isothiazolone.

In another embodiment the present invention provides a microbicidalcomposition comprising a synergistic mixture, the first component ofwhich is 2-methyl-3-isothiazolone, and the second component of which isone or more commercial microbicides selected from the group consistingof citric acid and benzyl alcohol; wherein the ratio of the firstcomponent to the second component is from 1/8 to 1/24 when the secondcomponent is citric acid; wherein the ratio of the first component tothe second component is from 1/0.13 to 1/32 or from 1/80 to 1/1600 whenthe second component is benzyl alcohol; and wherein the composition issubstantially free of halogenated 3-isothiazolone.

The present invention further provides a method of inhibiting the growthof microorganisms in a locus comprising introducing to, at or on, thelocus a microorganism inhibiting amount of the aforementionedsynergistic mixture; and wherein the amount of synergistic mixture isfrom 0.1 to 10,000 parts per million active ingredient.

In a preferred embodiment the present invention provides a method ofinhibiting the growth of microorganisms as described above wherein themicroorganisms are selected from one or more of bacterial and fungalmicroorganisms.

DETAILED DESCRIPTION

We have discovered that 2-methyl-3-isothiazolone (MI) may be combinedwith selected commercial microbicides to provide enhanced microbicidalefficacy at a combined active ingredient level lower than that of thecombined individual 3-isothiazolone or commercial microbicide effectiveuse levels, where the resulting composition is substantially free ofhalogenated 3-isothiazolone. Preferably the microbicidal compositionsare also substantially free of metal salt stabilizers, such as nitrateor magnesium salts; these “salt-free” microbicidal compositions areespecially useful to protect personal care compositions againstmicrobial contamination.

It has been discovered that mixtures of MI with one or more of thecommercial microbicides of the present invention in a ratio of 1/0.001to 1/1000 result in synergistic microbicidal activities against a widerange of microorganisms. Synergy occurs when the disruptive interactionon the organisms treated by the two compounds together is greater thanthe sum of such interactions of both compounds when used alone. Suchsynergy does not arise from the expected activity of the components whenadded together.

As used herein, the following terms have the designated definitions,unless the context clearly indicates otherwise. The term “microbicide”refers to a compound capable of inhibiting the growth of or controllingthe growth of microorganisms at a locus; microbicides includebactericides, fungicides and algaecides. The term “microorganism”includes, for example, fungi (such as yeast and mold), bacteria andalgae. The term “locus” refers to an industrial system or productsubject to contamination by microorganisms. The following abbreviationsare used throughout the specification: ppm=parts per million by weight(weight/weight), mL=milliliter, ATCC=American Type Culture Collection,and MIC=minimum inhibitory concentration. Unless otherwise specified,ranges listed are to be read as inclusive and combinable, temperaturesare in degrees centigrade (° C.), and references to percentages (%) areby weight. “Salt-free” means that the composition contains zero or up to0.5%, preferably zero or up to 0.1%, and more preferably zero or up to0.01%, of metal salt, based on weight of the composition.

The microbicidal compositions of the present invention are substantiallyfree of halogenated 3-isothiazolone; that is, zero or up to 3%,preferably zero or up to 1% and more preferably zero or up to 0.5%, ofhalogenated 3-isothiazolone may be present, based on combined weight ofhalogenated 3-isothiazolone and 2-methyl-3-isothiazolone. Microbicidalcompositions dependent on the presence of halogenated 3-isothiazoloneare susceptible to chemical degradation and may require additionalstabilizer components, such as the aforementioned metal saltstabilizers; salt stabilizers sometimes create unacceptable propertiesin finished formulations. For this reason it is desirable to providemicrobicide formulations substantially free of halogenated3-isothiazolone, but that still provide the degree of antimicrobialprotection provided by the halogenated 3-isothiazolones; such are themicrobicidal compositions of the present invention that are based on2-methyl-3-isothiazolone, which do not require metal stabilizers.

MI may be used in the synergistic mixtures of the present invention “asis” or may first be formulated with a solvent or a solid carrier.Suitable solvents include, for example, water; glycols, such as ethyleneglycol, propylene glycol, diethylene glycol, dipropylene glycol,polyethylene glycol, and polypropylene glycol; glycol ethers; alcohols,such as methanol, ethanol, propanol, phenethyl alcohol andphenoxypropanol; ketones, such as acetone and methyl ethyl ketone;esters, such as ethyl acetate, butyl acetate, triacetyl citrate, andglycerol triacetate; carbonates, such as propylene carbonate anddimethyl carbonate; and mixtures thereof. It is preferred that thesolvent is selected from water, glycols, glycol ethers, esters andmixtures thereof. Suitable solid carriers include, for example,cyclodextrin, silicas, diatomaceous earth, waxes, cellulosic materialsand charcoal. It is preferred that MI is formulated in water.

The second microbicide components of the combinations of the presentinvention are well-known and generally commercially availablemicrobicides, and include benzoic acid, citric acid, sorbic acid,1,2-dibromo-2,4-dicyanobutane, 1,3-dimethylol-5,5-dimethylhydantoin,phenoxyethanol, benzyl alcohol, zinc pyrithione and climbazole. Theratio of MI to the second microbicide component is typically from1/0.001 to 1/1000, and preferably from 1/0.05 to 1/100. Thesemicrobicides may be used in the synergistic mixtures of the presentinvention “as is” or may first be formulated together with a suitablesolvent, a solid carrier or as a dispersion. Suitable solvents and solidcarriers are those described above for MI.

When a water-insoluble second microbicide component is formulated in asolvent, the formulation may optionally contain surfactants. When suchformulations contain surfactants, they are generally in the form ofemulsive concentrates, emulsions, microemulsive concentrates, ormicroemulsions. Emulsive concentrates form emulsions upon the additionof a sufficient amount of water. Microemulsive concentrates formmicroemulsions upon the addition of a sufficient amount of water. Suchemulsive and microemulsive concentrates are generally well known in theart; it is preferred that such formulations are free of surfactants.U.S. Pat. No. 5,444,078 may be consulted for further general andspecific details on the preparation of various microemulsions andmicroemulsive concentrates.

Any formulation of MI may be used with any formulation of the secondmicrobicide component in the synergistic mixtures of the presentinvention. When both the MI and the second microbicide component areeach first formulated with a solvent, the solvent used for MI may be thesame as or different from the solvent used to formulate the othercommercial microbicide. It is preferred that the two solvents aremiscible. In the alternative, the MI and the other microbicide may becombined directly and then a solvent added to the mixture.

Those skilled in the art will recognize that the MI and the secondmicrobicide component of the present invention may be added to a locussequentially, simultaneously, or may be combined before being added tothe locus. It is preferred that the MI and the second microbicidecomponent be added to a locus simultaneously or combined prior to beingadded to the locus. When the microbicides are combined prior to beingadded to a locus, such combination may optionally contain adjuvants,such as, for example, solvent, thickeners, anti-freeze agents,colorants, sequestrants (such as ethylenediamine-tetraacetic acid,ethylenediaminedisuccinic acid, iminodisuccinic acid and salts thereof),dispersants, surfactants, stabilizers, scale inhibitors andanti-corrosion additives.

The microbicidal compositions of the present invention can be used toinhibit the growth of microorganisms by introducing a microbicidallyeffective amount of the compositions onto, into, or at a locus subjectto microbial attack. Suitable loci include, for example: cooling towers;air washers; boilers; mineral slurries; wastewater treatment; ornamentalfountains; reverse osmosis filtration; ultrafiltration; ballast water;evaporative condensers; heat exchangers; pulp and paper processingfluids; plastics; emulsions; dispersions; paints; latexes; coatings,such as varnishes; construction products, such as mastics, caulks, andsealants; construction adhesives, such as ceramic adhesives, carpetbacking adhesives, and laminating adhesives; industrial or consumeradhesives; photographic chemicals; printing fluids; household products,such as bathroom and kitchen cleaners; cosmetics; toiletries; shampoos;soaps; detergents; industrial cleaners; floor polishes; laundry rinsewater; metalworking fluids; conveyor lubricants; hydraulic fluids;leather and leather products; textiles; textile products; wood and woodproducts, such as plywood, chipboard, flakeboard, laminated beams,oriented strandboard, hardboard, and particleboard; petroleum processingfluids; fuel; oilfield fluids, such as injection water, fracture fluids,and drilling muds; agriculture adjuvant preservation; surfactantpreservation; medical devices; diagnostic reagent preservation; foodpreservation, such as plastic or paper food wrap; pools; and spas.

Preferably, the microbicidal compositions of the present invention areused to inhibit the growth of microorganisms at a locus selected fromone or more of emulsions, dispersions, paints, latexes, householdproducts, cosmetics, toiletries, shampoos, soaps, detergents andindustrial cleaners. In particular, the microbicidal compositions areuseful in personal care applications, such as hair care (for example,shampoo and dyes) and skin care (for example, sunscreens, cosmetics,soaps, lotions and creams) formulations.

When the synergistic compositions of the present invention are used inpersonal care compositions, the formulated compositions may alsocomprise one or more ingredients selected from UV radiation-absorbingagents, surfactants, rheology modifiers or thickeners, fragrances,moisturizers, humectants, emollients, conditioning agents, emulsifiers,antistatic aids, pigments, dyes, tints, colorants, antioxidants,reducing agents and oxidizing agents.

Suitable UV radiation-absorbing agents (including chemical absorbers andphysical blockers) include, for example, oxybenzone, dioxybenzone,sulisobenzone, menthyl anthranilate, para-aminobenzoic acid, amylpara-dimethylaminobenzoic acid, octyl para-dimethylaminobenzoate, ethyl4-bis(hydroxypropyl)aminobenzoate, polyethylene glycol (PEG-25)para-aminobenzoate, diethanolamine para-methyoxycinna mate,2-ethoxyethyl para-methoxycinnamate, ethylhexyl para-methoxycinnamate,octyl para-methoxycinnamate, isoamyl para-methoxycinnamate,2-ethylhexyl-2-cyano-3,3-diphenylacrylate, 2-ethylhexyl salicylate,homomenthyl salicylate, glyceryl aminobenzoate, triethanolaminesalicylate, digalloyl trioleate, lawsone with dihydroxyacetone,2-phenylbenzimidazole-5-sulfonic acid, 4-methylbenzylidine camphor,avobenzone, titanium dioxide and zinc oxide. Alternatively, UVradiation-absorbing agents such as triazines, benzotriazoles, vinylgroup-containing amides, cinnamic acid amides and sulfonatedbenzimidazoles may also be used.

Suitable surfactants include, for example, nonionic, anionic, cationicand amphoteric surfactants and mixtures thereof; such as PPG 28 Buteth35, PEG 75 lanolin, perfluoropolymethyl isopropyl ether, octoxynol-9,PEG-25 hydrogenated castor oil, polyethylene terephthalate, polyethyleneglycol 25 glyceryl trioleate, oleth-3 phosphate, PPG-5-ceteth-10phosphate, PEG-20 methyl glucose ether, glycereth-7-triacetate andN-alkyl substituted lactam (such as N-octyl pyrrolidone).

Suitable thickeners or rheology modifiers include, for example,hydrophobically modified nonionic ethoxylated urethanes, polycarboxylicacid thickeners such as acrylates/steareth-20 methacrylate copolymer,carbomers, acrylates copolymer and acrylates C₁₋₃₀ alkyl acrylatecroespolymer.

The personal care compositions improved by the method of this inventioninclude, for example:

(a) hair care formulations, including shampoos, hair dyes, hairconditioners, gels, mousses and hair sprays; and

(b) skin care and nail care formulations, including nail coatings,cosmetics, astringents, depilatories, facial make-up formulations,sunscreens and sunblocks, premoistened wipes, hand creams, hand and bodysoaps, and hand and body lotions.

The cosmetic formulations typically contain water, film formingmaterials, emulsifiers, softeners, emollients, oils, stabilizers,thickeners, neutralizers, perfume, colorants, pigments and combinationsthereof. The sunscreen formulations typically contain UVradiation-absorbing agents, water, film forming materials, emulsifiers,emollients, waterproofing agents, oils, stabilizers, thickeners,preservatives, perfume, colorants, insecticides, humectants andcombinations thereof.

Optionally, other additives, such as additional film forming agents,plasticizers, antifoaming agents, leveling aids, excipients, vitamins,natural extracts, proteins, sequestrants, dispersants, antioxidants,suspending agents and solvents may be added to the personal careformulations described above. Suitable solvents include, for example,C₁-C₁₂ straight or branched chain alcohols such as ethanol, isopropanol,or propanol; alkyl esters such as ethyl acetate; ketones; andcombinations thereof.

The specific amount of the synergistic combinations necessary to inhibitor control the growth of microorganisms in a locus depends upon theparticular compounds in the combination and particular locus to beprotected. Typically, the amount of the synergistic combinations of thepresent invention to control the growth of microorganisms in a locus issufficient if it provides from 0.1 to 10,000 ppm active ingredient ofthe synergistic mixture in the locus. It is preferred that thesynergistic mixture be present in an amount of 0.5 to 5000 ppm, and morepreferably from 1 to 3000 ppm, in the locus.

Microorganisms evaluated for effectiveness of control by thecompositions of the present invention include bacterial (P. aeruginosaand S. aureus) and fungal (A. niger and C. albicans) microorganisms, inparticular Gram (−) bacteria and Gram (+) bacteria, as well as yeast andmold.

Combinations of MI with sorbic acid, 1,2-dibromo-2,4-dicycanobutane,1,3-dimethylol-5,5-dimethylhydantoin or phenoxyethanol were particularlyeffective against both Gram (−) and Gram (+) bacteria. Combinations ofMI with benzoic acid, zinc pyrithione or benzyl alcohol wereparticularly effective against Gram (−) bacteria. Combinations of MIwith citric acid or climbazole were particularly effective against Gram(+) bacteria. Combinations of MI with phenoxyethanol, zinc pyrithione orbenzyl alcohol were particularly effective against both yeast and mold.Combinations of MI with benzoic acid, 1,2-dibromo-2,4-dicycanobutane orclimbazole were particularly effective against yeast. Combinations of MIwith sorbic acid were particularly effective against mold. Combinationsof MI with benzoic acid, sorbic acid, 1,2-dibromo-2,4-dicycanobutane,phenoxyethanol, zinc pyrithione, climbazole or benzyl alcohol wereeffective against both bacterial and fungal microorganisms.

When benzoic acid is used in the synergistic mixtures of the presentinvention, it is preferred that the ratio of MI to benzoic acid is from1/0.1 to 1/100 and more preferably from 1/0.13 to 1/67. When citric acidis used in the synergistic mixtures of the present invention, it ispreferred that the ratio of MI to citric acid is from 1/8 to 1/40 andmore preferably from 1/8 to 1/24. When sorbic acid is used in thesynergistic mixtures of the present invention, it is preferred that theratio of MI to sorbic acid is from 1/2 to 1/150, more preferably from1/4 to 1/133 and most preferably from 1/4 to 1/67. When1,2-dibromo-2,4-dicyanobutane is used in the synergistic mixtures of thepresent invention, it is preferred that the ratio of MI to1,2-dibromo-2,4-dicyanobutane is from 1/0.4 to 1/100. When1,3-dimethylol-5,5-dimethylhydantoin is used in the synergistic mixturesof the present invention, it is preferred that the ratio of MI to1,3-dimethylol-5,5-dimethylhydantoin is from 1/0.05 to 1/100 and morepreferably from 1/0.06 to 1/80. When phenoxyethanol is used in thesynergistic mixtures of the present invention, it is preferred that theratio of MI to phenoxyethanol is from 1/1 to 1/1000 and more preferablyfrom 1/2 to 1/800. When zinc pyrithione is used in the synergisticmixtures of the present invention, it is preferred that the ratio of MIto zinc pyrithione is from 1/0.001 to 1/20 and more preferably from1/0.0013 to 1/13. When climbazole is used in the synergistic mixtures ofthe present invention, it is preferred that the ratio of MI toclimbazole is from 1/0.03 to 1/30 and more preferably from 1/0.05 to1/24. When benzyl alcohol is used in the synergistic mixtures of thepresent invention, it is preferred that the ratio of MI benzyl alcoholis from 1/0.13 to 1/32 or from 1/80 to 1/1600 and more preferably from1/80 to 1/400.

Some embodiments of the invention are described in detail in thefollowing Examples. All ratios, parts and percentages are expressed byweight unless otherwise specified, and all reagents used are of goodcommercial quality unless otherwise specified. Abbreviations used in theExamples and Tables are listed below with the correspondingdescriptions:

-   -   MI=2-methyl-3-isothiazolone    -   SI=synergy index    -   MIC=minimum inhibitory concentration

Examples

The synergism of the combinations of the present invention wasdemonstrated by testing a wide range of concentrations and ratios of thecompounds.

Synergism was determined by an industrially accepted method described byKull, F. C.; Eisman, P. C.; Sylwestrowicz, H. D. and Mayer, R. L., inApplied Microbiology 9:538-541 (1961), using the ratio determined by theformula:

Q _(a) /Q _(A) +Q _(b) /Q _(B)=Synergy Index (“SI”)

wherein:

-   -   Q_(A)=concentration of compound A (first component) in ppm,        acting alone, which produced an end point (MIC of Compound A).    -   Q_(a)=concentration of compound A in ppm, in the mixture, which        produced an end point.

Q_(B)=concentration of compound B (second component) in ppm, actingalone, which produced an end point (MIC of Compound B).

Q_(b)=concentration of compound B in ppm, in the mixture, which producedan end point.

When the sum of Q_(a)/Q₁ and Q_(b)/Q_(B) is greater than one, antagonismis indicated. When the sum is equal to one, additivity is indicated, andwhen less than one, synergism is demonstrated. The lower the SI, thegreater the synergy shown by that particular mixture. The minimuminhibitory concentration (MIC) of a microbicide is the lowestconcentration tested under a specific set of conditions that preventsthe growth of added microorganisms.

Synergy tests were conducted using standard microtiter plate assays withmedia designed for optimal growth of the test microorganism. Minimalsalt medium supplemented with 0.2% glucose and 0.1% yeast extract (M9GYmedium) was used for testing bacteria; Potato Dextrose Broth (PDBmedium) was used for testing yeast and mold. In this method, a widerange of combinations of microbicides was tested by conducting highresolution MIC assays in the presence of various concentrations of MI.High resolution MICs were determined by adding varying amounts ofmicrobicide to one column of a microtitre plate and doing subsequentten-fold dilutions using an automated liquid handling system to obtain aseries of endpoints ranging from 2 ppm to 10,000 ppm active ingredient.The synergy of the combinations of the present invention was determinedagainst two bacteria, Staphylococcus aureus (S. aureus—ATCC #6538) orPseudomonas aeruginosa (P. aeruginosa—ATCC #9027), a yeast, Candidaalbicans (C. albicans—ATCC 1.0231), and a mold, Aspergillus niger (A.niger—ATCC 16404). The bacteria were used at a concentration of about5×10′ bacteria per mL and the yeast and mold at 5×105 fungi per mL.These microorganisms are representative of natural contaminants in manyconsumer and industrial applications. The plates were visually evaluatedfor microbial growth (turbidity) to determine the MIC after variousincubation times at 25° C. (yeast and mold) or 30° C. (bacteria).

The test results for demonstration of synergy of the microbicidecombinations of the present invention are shown below in Tables 1through 9. In each test, First Component (A) was MI and the SecondComponent (B) was the other commercial microbicide. Each table shows thespecific combinations of MI and the second component; results againstthe microorganisms tested with incubation times; the end-point activityin ppm measured by the MIC for MI alone (Q_(A)), for the secondcomponent alone (Q_(B)), for MI in the mixture (Q_(a)) and for secondcomponent in the mixture (Q_(b)); the calculated SI value; and the rangeof synergistic ratios for each combination tested (MI/second componentor A/B).

TABLE 1 First Component (A) = 2-Methyl-3-isothiazolone Second Component(B) = benzoic acid Microorganism Q_(a) Q_(b) SI A/B A. niger 0 100001.00 — (1 week) 50 10000 1.17 1/200 200 0 1.67 1/50 300 0 1.00 — C.albicans 0 5000 1.00 — (48 hours) 125 2000 1.03 1/16 125 1000 0.83 1/8125 800 0.79 1/6.4 125 600 0.75 1/4.8 125 500 0.73 1/4 125 400 0.711/3.2 125 300 0.69 1/2.4 125 200 0.67 1/1.6 125 100 0.65 1/0.8 125 800.64 1/0.64 125 60 0.64 1/0.48 125 50 0.64 1/0.4 125 40 0.63 1/0.32 15030 0.76 1/0.2 150 20 0.75 1/0.13 200 0 1.00 — S. aureus 0 6000 1.00 —(72 hours) 15 6000 1.25 1/400 50 5000 1.67 1/100 60 0 1.00 — P.aeruginosa 0 6000 1.00 — (72 hours) 15 2000 1.08 1/133 15 1000 0.92 1/6715 800 0.88 1/53 15 600 0.85 1/40 15 500 0.83 1/33 15 400 0.82 1/27 15300 0.80 1/20 20 0 1.00 —

The synergistic ratios of MI/benzoic acid range from 1/0.13 to 1/67. TheMI/benzoic acid combination showed enhanced control of Gram (−) bacteriaand yeast.

TABLE 2 First Component = 2-methyl-3-isothiazolone Second Component =citric acid Microorganism Q_(a) Q_(b) SI A/B A. niger 0 10000 1.00 — (1week) 50 10000 1.17 1/200 200 10000 1.67 1/50 300 0 1.00 — C. albicans 010000 1.00 — (72 hours) 50 10000 1.25 1/200 150 10000 1.75 1/67 200 01.00 — S. aureus 0 4000 1.00 — (24 hours) 15 3000 1.13 1/200 25 20001.13 1/80 25 1000 0.88 1/40 25 800 0.83 1/32 25 600 0.78 1/24 25 5000.75 1/20 25 400 0.73 1/16 25 300 0.70 1/12 25 200 0.68 1/8 40 0 1.00 —P. aeruginosa 0 6000 1.00 — (48 hours) 2.5 6000 1.13 1/2400 15 5000 1.581/333 20 0 1.00 —

The synergistic ratios of MI/citric acid range from 1/8 to 1/40,preferably from 1/8 to 1/24. The MI/citric acid combinations showenhanced control of Gram (+) bacteria.

TABLE 3 Compound A = 2-methyl-3-isothiazolone Compound B = sorbic acidMicroorganism Q_(a) Q_(b) SI A/B A. niger 0 4000 1.00 — (96 hours) 503000 0.92 1/60 75 3000 1.00 1/40 75 2000 0.75 1/27 100 3000 1.08 1/30100 2000 0.83 1/20 125 3000 1.17 1/24 125 2000 0.92 1/16 125 1000 0.671/8 150 2000 1.00 1/13 150 1000 0.75 1/6.7 150 800 0.70 1/5 200 20001.17 1/10 200 1000 0.92 1/5 200 800 0.87 1/4 300 0 1.00 — C. albicans 0400 1.00 — (72 hours) 50 400 1.25 1/8 150 300 1.50 1/2 200 0 1.00 — S.aureus 0 10000 1.00 (72 hours) 40 4000 1.07 1/100 40 3000 0.97 1/75 502000 1.03 1/40 50 1000 0.93 1/20 50 800 0.91 1/16 50 600 0.89 1/12 50500 0.88 1/10 50 400 0.87 1/8 50 300 0.86 1/6 50 200 0.85 1/4 60 0 1.00— P. aeruginosa 0 10000 1.00 — (48 hours) 15 3000 1.05 1/200 15 20000.95 1/133 15 1000 0.85 1/67 20 0 1.00 —

The synergistic ratios of MI/sorbic acid range from 1/4 to 1/133,preferably from 1/4 to 1/67. The MI/sorbic acid combination showedenhanced control of mold and both Gram (+) and Gram (−) bacteria.

TABLE 4 First Component = 2-methyl-3-isothiazolone Second Component =1,2-dibromo-2,4-dicyanobutane Microorganism Q_(a) Q_(b) SI A/B A. niger0 100 1.00 — (1 week) 50 100 1.17 1/2 200 100 1.67 1/0.5 300 0 1.00 — C.albicans 0 500 1.00 — (72 hours) 125 200 1.03 1/1.6 150 200 1.15 1/1.3150 100 0.95 1/0.7 150 80 0.91 1/0.5 150 60 0.87 1/0.4 200 0 1.00 — S.aureus 0 1000 1.00 — (72 hours) 40 800 1.47 1/20 50 200 1.03 1/4 50 1000.93 1/2 50 80 0.91 1/1.6 60 0 1.00 — P. aeruginosa 0 800 1.00 — (24hours) 5 500 0.96 1/100 7.5 400 1.00 1/53 7.5 300 0.88 1/40 10 300 1.041/30 10 200 0.92 1/20 10 100 0.79 1/10 15 0 1.00 —

The synergistic ratios of MI/1,2-dibromo-2,4-dicyanobutane range from1/0.4 to 1/100. The MI/1,2-dibromo-2,4-dicyanobutane combination showedenhanced control of yeast and both Gram (+) and Gram (−) bacteria.

TABLE 5 First Component = 2-methyl-3-isothiazolone Second Component =1,3-dimethylol-5,5-dimethylhydantoin Microorganism Q_(a) Q_(b) SI A/B A.niger 0 3000 1.00 — (1 week) 50 3000 1.17 1/60 200 2000 1.33 1/10 300 01.00 — C. albicans 0 3000 1.00 — (72 hours) 50 3000 1.25 1/60 150 30001.75 1/20 200 0 1.00 — S. aureus 0 200 1.00 — (72 hours) 15 100 0.751/6.7 15 80 0.65 1/5.3 15 60 0.55 1/4 25 100 0.92 1/4 25 80 0.82 1/3.225 60 0.72 1/2.4 25 50 0.67 1/2 40 80 1.07 1/2 40 60 0.97 1/1.5 40 500.92 1/1.25 40 40 0.67 1/1 40 30 0.82 1/0.75 50 40 1.03 1/0.8 50 30 0981/0.6 50 20 0.93 1/0.4 50 10 0.88 1/0.2 50 5 0.86 1/0.1 50 3 0.85 1/0.0660 0 1.00 — P. aeruginosa 0 300 1.00 — (24 hours) 2.5 200 0.83 1/80 5200 1.00 1/40 5 100 0.67 1/20 5 80 0.60 1/16 7.5 200 1.17 1/27 7.5 1000.83 1/13 7.5 80 0.77 1/11 7.5 60 0.70 1/8 7.5 50 0.67 1/6.7 10 100 1.001/10 10 80 0.93 1/8 10 60 0.87 1/6 10 40 0.80 1/4 10 20 0.73 1/2 10 80.69 1/0.8 15 0 1.00 —

The synergistic ratios of MI/1,3-dimethylol-5,5-dimethylhydantoin rangefrom 1/0.06 to 1/80. The MI/1,3-dimethylol-5,5-dimethylhydantoincombination showed enhanced control of both Gram (+) and Gram (−)bacteria.

TABLE 6 First Component = 2-methyl-3-isothiazolone Second Component =phenoxyethanol Microorganism Q_(a) Q_(b) SI A/B A. niger 0 4000 1.00 —(72 hours) 50 3000 0.92 1/60 50 2000 0.67 1/40 75 3000 1.00 1/40 75 20000.75 1/27 100 3000 1.08 1/30 100 2000 0.83 1/20 125 3000 1.17 1/24 1252000 0.92 1/16 150 2000 1.00 1/13 300 0 1.00 — C. albicans 0 4000 1 00 —(72 hours) 50 3000 1.00 1/60 75 3000 1.13 1/40 75 2000 0.88 1/27 1002000 1.00 1/20 125 2000 1.13 1/16 125 1000 0.88 1/8 125 800 0.83 1/6.4125 600 0.78 1/4.8 125 500 0.75 1/4 130 1000 1.00 1/6.7 130 800 0.951/5.3 130 600 0.90 1/4 150 300 0.38 1/3.3 150 300 0.83 1/2 200 0 1.00 —S. aureus 0 4000 1.00 — (72 hours) 50 2000 1.13 1/40 60 1000 1.00 1/1760 800 0.95 1/13 80 0 1.00 — P. aeruginosa 0 3000 1.00 — (24 hours) 2.52000 0.83 1/800 5 2000 1.00 1/400 7.5 2000 1.17 1/267 7.5 1000 0.831/133 7.5 800 0.77 1/107 7.5 600 0.70 1/80 10 1000 1.00 1/100 10 8000.93 1/80 10 600 0.87 1/60 10 400 0.80 1/40 10 300 0.77 1/30 15 0 1.00 —

The synergistic ratios of MI/phenoxyethanol range from 1/2 to 1/800. TheMI/phenoxyethanol combination showed enhanced control of Gram (+) andGram (−) bacteria as well as yeast and mold.

TABLE 7 First Component = 2-methyl-3-isothiazolone Second Component =zinc pyrithione Microorganism Q_(a) Q_(b) SI A/B A. niger 0 80 1.00 — (1week) 50 10 0.29 1/0.2 75 60 1.00 1/0.8 75 50 0.88 1/0.67 75 40 0.751/0.53 75 30 0.63 1/0.4 75 20 0.50 1/0.27 75 10 0.38 1/0.13 75 8 0.351/0.1 100 60 1.08 1/0.6 100 50 0.96 1/0.5 100 40 0.83 1/0.4 100 30 0.711/0.3 100 20 0.58 1/0.2 100 10 0.46 1/0.1 125 50 1.04 1/0.4 125 40 0.921/0.32 125 30 0.79 1/0.24 125 20 0.67 1/0.16 125 8 0.52 1/0.06 150 401.00 1/0.27 150 30 0.88 1/0.2 150 20 0.75 1/0.13 150 8 0.60 1/0.05 20030 1.04 1/0.15 200 20 0.92 1/0.1 200 10 0.79 1/0.05 200 6 0.74 1/0.03300 0 1.00 — C. albicans 0 80 1.00 — (48 hours) 50 60 1.00 1/1.2 50 500.88 1/1 50 40 0.75 1/0.8 75 50 1.00 1/0.67 75 40 0.88 1/0.53 75 30 0.751/0.4 75 20 0.63 1/0.27 75 8 0.48 1/0.1 100 40 1.00 1/0.4 100 30 0.881/0.3 100 20 0.75 1/0.2 100 10 0.63 1/0.1 100 6 0.58 1/0.06 125 30 1.001/0.24 125 20 0.88 1/0.16 125 10 0.75 1/0.08 125 5 0.69 1/0.04 125 30.66 1/0.024 150 20 1.00 1/0.13 150 10 0.88 1/0.067 150 5 0.81 1/0.033150 1 0.76 1/0.0067 150 0.5 0.76 1/0.0033 150 0.2 0.75 1/0.0013 200 01.00 — S. aureus 0 20 1.00 — (72 hours) 15 20 1.30 1/1.3 40 20 1.801/0.5 50 0 1.00 — P. aeruginosa 0 200 1.00 — (72 hours) 7.5 100 0.881/13 10 100 1.00 1/10 10 80 0.90 1/8 15 50 1.00 1/3.3 15 40 0.95 1/2.715 30 0.90 1/2 15 20 0.85 1/1.3 15 10 0.80 1/0.67 15 5 0.78 1/0.33 15 30.77 1/0.2 20 0 1.00 —

The synergistic ratios of MI/zinc pyrithione range from 1/0.0013 to1/13. The MI/zinc pyrithione combination showed enhanced control of Gram(−) bacteria and both yeast and mold.

TABLE 8 First Component = 2-methyl-3-isothiazolone Second Component =climbazole Microorganism Q_(a) Q_(b) SI A/B C. albicans 0 300 1.00 — (72hours) 50 200 0.92 1/4 75 200 1.04 1/2.7 75 100 0.71 1/1.3 75 60 0.581/0.8 75 40 0.51 1/0.53 100 200 1.17 1/2 100 100 0.83 1/1 100 80 0.771/0.8 100 60 0.70 1/0.6 100 40 0.63 1/0.4 100 20 0.57 1/0.2 125 200 1.291/1.6 125 100 0.96 1/0.8 125 80 0.89 1/0.64 125 50 0.79 1/0.4 125 300.73 1/0.24 125 10 0.66 1/0.08 150 80 1.02 1/0.53 130 60 0.95 1/0.4 15040 0.88 1/0.27 150 20 0.82 1/0.13 150 8 0.78 1/0.053 200 0 1.00 — S.aureus 0 2000 1.00 — (24 hours) 25 800 1.03 1/32 25 600 0.93 1/24 40 01.00 —

The synergistic ratios of MI/climbazole range from 1/0.05 to 1/24. TheMI/climbazole combination showed enhanced control of Gram (+) bacteriaand yeast.

TABLE 9 First Component = 2-methyl-3-isothiazolone Second Component =benzyl alcohol Microorganism Q_(a) Q_(b) SI A/B A. niger 0 8000 1.00 —(72 hours) 100 6000 1.08 1/60 125 5000 1.04 1/40 125 4000 0.92 1/32 1253000 0.79 1/24 300 0 1.00 — C. albicans 0 6000 1.00 — (48 hours) 1003000 1.00 1/30 125 3000 1.13 1/24 125 2000 0.96 1/16 125 1000 0.79 1/8125 600 0.73 1/4.8 125 400 0.69 1/3.2 125 200 0.66 1/1.6 125 80 0.641/0.64 150 20 0.75 1/0.13 150 2000 1.08 1/13 150 1000 0.92 1/6.7 150 8000.88 1/5.3 150 500 0.83 1/3.3 150 200 0.78 1/1.3 150 80 0.76 1/0.53 15040 0.76 1/0.27 200 0 1.00 — S. aureus 0 8000 1.00 — (72 hours) 40 40001.17 1/100 50 2000 1.08 1/40 50 1000 0.96 1/20 50 800 0.93 1/16 50 5000.90 1/10 50 300 0.87 1/6 50 100 0.85 1/2 50 50 0.84 1/1 60 0 1.00 — P.aeruginosa 0 6000 1.00 — (24 hours) 2.5 5000 1.00 1/2000 2.5 4000 0.831/1600 5 4000 1.00 1/800 5 3000 0.83 1/600 5 2000 0.67 1/400 5 800 0.471/160 7.5 2000 0.83 1/267 7.5 1000 0.67 1/133 7.5 600 0.60 1/80 10 20001.00 1/200 10 1000 0.83 1/100 10 800 0.80 1/80 10 500 0.75 1/50 10 3000.72 1/30 10 100 0.68 1/10 10 60 0.68 1/6 15 0 1.00 —

The synergistic ratios of MI/benzyl alcohol range from 1/0.1 to 1/1600,preferably from 1/0.13 to 1/32 or from 1/80 to 1/1600, and morepreferably from 1/80 to 1/400. The MI/benzyl alcohol combination showedenhanced control of Gram (+) and Gram (−) bacteria as well as mold andyeast.

What is claimed is: 1-10. (canceled)
 11. A microbicidal compositionconsisting of 2-methyl-3-isothiazolone and zinc pyrithione which iseffective for inhibiting growth of a microorganism selected from a mold,a yeast or a bacteria, wherein either (a) the ratio of2-methyl-3-isothiazolone to zinc pyrithione is from 1/0.03 to 1/0.67,and over such range the composition exhibits synergy for inhibitinggrowth of Aspergillus niger; (b) the ratio of 2-methyl-3-isothiazoloneto zinc pyrithione is from 1/0.0013 to 1/0.67, and over such range thecomposition exhibits synergy for inhibiting growth of Candida albicans;or (c) the ratio of 2-methyl-3-isothiazolone to zinc pyrithione is from110.2 to 1/2.7 and over such range the composition exhibits synergy forinhibiting growth of Pseudomonas aeruginosa.
 12. The microbicidalcomposition of claim 11, wherein the composition is effective forinhibiting growth of both Aspergillus niger and Candida albicans,wherein the ratio of 2-methyl-3-isothiazolone to zinc pyrithione is from1/0.16 to 1/0.67, and over the range of 1/0.16 to 1/0.67 the compositionexhibits synergy for inhibiting such growth.
 13. The microbicidalcomposition of claim 11, wherein the composition is effective forinhibiting growth of both Aspergillus niger, Candida albicans, andPseudomonas aeruginosa, wherein the ratio of 2-methyl-3-isothiazolone tozinc pyrithione is from 1/0.2 to 1/0.67, and over the range of 1/0.2 to1/0.67 the composition exhibits synergy for inhibiting such growth.